Abstract
To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochrome c in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochrome c-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5'-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.
Highlights
Caspases, a highly conserved family of cysteine proteases that cleave their substrates after an aspartate residue, play fundamental roles in the initiation and execution of apoptosis
We provide evidence that cytochrome c and dATP are required to promote oligomerization of Apaf-1 and that mature caspase-9 is released from the full-length Apaf-1 apoptosome but not from the truncated Apaf-530 complex
The human Apaf-1L is similar in structure to the recently cloned mouse Apaf-1, which has an additional WD-40 repeat [13]
Summary
A highly conserved family of cysteine proteases that cleave their substrates after an aspartate residue, play fundamental roles in the initiation and execution of apoptosis (reviewed in Refs. 1– 4). The Apaf-1cytochrome c complex recruits procaspase-9 in a dATP/ ATP-dependent manner through a CARD-CARD1 interaction, resulting in its activation and presumably the release of mature caspase-9 from the apoptosome [5, 9].
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